A SLC6 transporter of the novel B0,- system aids in absorption and detection of nutrient amino acids in Caenorhabditis elegans

Abstract

Summary Nutrient Amino acid Transporters (NATs) of SoLute Carrier family 6 (SLC6) mediate uptake of essential amino acids in mammals and insects. Phylogenomic analysis of the Caenorhabditis elegans (Ce) SLC6 family identifies 5 genes paralogous to an insect-specific NAT subfamily. Here we cloned and characterized the first representative of the identified nematode-specific transporters, SNF-5. SNF-5 mediates broad spectrum cation-coupled transport of neutral amino acids with submillimolar affinities and stoichiometry of 1AA:1Na+, except for 1L-Pro:2 Na+. Unexpectedly, it transports acidic L-Glu- and L-Asp- (AA -1:3Na+), revealing it to be the first member of a new B0,- system among characterized SLC6 transporters. This activity correlates with a unique positively charged His+ 377 in the substrate-binding pocket. snf-5 promoter-driven EGFP labels intestinal cells INT1-9 and three pairs of amphid sensory neurons: ASI, ADF, and ASK. These cells are intimately involved in control of dauer diapause, development, metabolism, and longevity. The snf-5 deletion mutants do not show apparent morphological disorders, but increase dauer formation while reducing dauer maintenance upon starvation. Overall, our studies characterized the first nematode-specific NAT and revealed important structural and functional aspects of this transporter. In addition to the predictable role in alimentary amino acid absorption, our results indicate possible neuronal roles of SNF-5 as an amino acid provider to specific neuronal functions, including sensing of amino acid availability.

Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation

“…In insects, amino acids taken up from midgut need to traverse at least three membranes to reach their intracellular site of use: (1) uptake into an epithelial gut cell, (2) basolateral efflux from the gut cell into the hemolymph, and (3) uptake into somatic cell. In general, SLC6 transporters potentially mediate the uptake steps (1) and (3), whereas other SLC members are involved in the efflux step (2) 1 39 .…”

“…In this survey, tissue expression profiles revealed that LdNAT1 mRNA levels were high or moderate in the larval gut, and lower in other surveyed tissues. Similarly, NAT genes in mammalians, other insect species and nematodes are highly expressed in the apical membranes of the alimentary canal, as well as in other organs with elevated requirements for essential amino acids 1 11 12 13 37 39 . For example, MsKAAT1 in M. sexta 7 , AeAAT1 11 and AeNAT5 14 in A. aegypti , AgNAT6 37 and AgNAT8 12 in A. gambiae were highly expressed in the larval guts.…”

A novel eukaryotic Na+ methionine selective symporter is essential for mosquito development

“…5B) emphasizes Na + binding at a fixed, nearly saturated, amino acid concentration. NATs preferably using 2:1 and 3:1 coupling stoichiometry (Metzler et al, 2013), and even triggering between 1 and 2 Na + ratios (Meleshkevitch et al, 2009), have been reported previously. The crystalized bacterial LeuT possesses two Na + binding sites (Yamashita et al, 2005) with distinct ion binding mechanisms (Noskov and Roux, 2008) that are additionally modulated through the binding of the transporter by organic substrates and Cl − .…”

“…The identified pattern of transcription suggests broad involvement of Ae NAT5 in alimentary and neuronal functions. However, the significance of these contributions was enigmatic because the biological role of NATs has never been experimentally assessed in insects, nor has it been systematically addressed in other model organisms beyond reported links of several human NAT-SLC6 members contributing to an aminoacidurias complex (Broer and Palacin, 2011) and the recent demonstration of a role for C. elegans SNF-5 in nematode development (Metzler et al, 2013). …”

“…7D, E, F). Recent studies of SNF-5, a broad spectra NAT with particularly high affinity to L-Met in a model of basal Ecdysozoa, C. elegans , has shown specific expression of this transporter in identifiable Amphid Sensory neurons: ASI, ASK, and ADF (Metzler et al, 2013). These cells detect amino acids and control worm development and metabolism.…”